We demonstrated that gene silencing of SOD2 in differentiated RGC-5 cells resulted in increased superoxide levels which likely caused decreased viability. These results were seen in the absence of glia or other retinal neurons, i.e. were cell-type autonomous. Effects on viability were seen in cells exposed to menadione, which increases levels of extramitochondrial superoxide (Meany et al., 2007
). The transfection efficiency of SOD2 siRNA was high, and the effects of SOD2 silencing began as early as 24 hours after transfection, with the most pronounced silencing at 72 hours after transfection.
Retinal ganglion cells are the primary cell type involved in optic neuropathies. They can be studied in vitro
in primary cell cultures of either mixed retinal cells or affinity-purified RGCs (Barres et al., 1988
). Purities of 95-99% are achievable, but require the binding of an antibody to cell-surface Thy-1, which may have other biological effects (Leifer et al., 1984
). If large numbers are RGCs are required, a correspondingly large number of animals need to be used. The RGC-5 cell line is an alternative (Krishnamoorthy et al., 2001
). RGC-5 cells are neuronal precursor cells that are mitotically active and do not have a neuronal morphology in their basal state. We previously showed that nanomolar concentrations of the broad-spectrum kinase inhibitor staurosporine induces RGC-5 differentiation, manifested by arrest of cellular proliferation, extension of dendrites and axons, and development of ionic channels similar to those seen in mature RGCs (Frassetto et al., 2006
; Lieven et al., 2007
). Others have reproduced the ability of staurosporine to differentiate RGC-5 cells (Harvey and Chintala, 2007
), although the question of whether they are truly RGCs or not is controversial, and therefore our results cannot necessarily be extrapolated to RGCs in vivo
(Van Bergen et al., 2009
). All experiments with RGC-5 cells were performed at a passage number of less than 8, to maintain their ability to differentiate into neurons. Although differentiated RGC-5 cells are not true RGCs, their homogeneity and purity make them useful for gene silencing experiments. They are also helpful for assess whether a biological mechanism in neurons is cell-type autonomous because they can be purified to homogeneity.
It is therefore possible that there are unrecognized differences between differentiated RGC-5 cells and RGCs. On the other hand, the fact that the current findings support the in vivo
results of Qi and colleagues in RGCs (2003)
is encouraging. Studying RGCs from mice in which SOD2 was inactivated by homologous recombination is not an alternative because homozygotes die within the first 10 days of life with a dilated cardiomyopathy, accumulation of lipid in liver and skeletal muscle, and metabolic acidosis (Li et al., 1995
). This indicates that SOD2 is required for normal biologic function of tissues by maintaining the integrity of mitochondrial enzymes susceptible to direct inactivation by superoxide. Treatment with an SOD mimetic, MnTBAP, rescued SOD2−/−
mutant mice and dramatically prolonged their survival (Melov et al., 2001
). Interestingly, animals developed a pronounced movement disorder progressing to total debilitation by 3 weeks of age. Future studies could include the use of purified RGC cultures from mice conditionally deficient in one or more SOD isoforms.
Two groups have used siRNA-mediated gene silencing in RGCs in vivo (Lingor et al., 2005
) and in RGC-5 cells against c-Jun, Bax and Apaf-1 (Lingor et al., 2005
), and PKR (Shimazawa et al., 2007
). However, those studies were performed with either undifferentiated cells or cells treated with concanavalin A, which does not produce a true neuronal morphology. The study of Lingor and colleagues (2005)
achieved RNA interference with custom single oligonucleotide duplexes, while we used pools of 4 duplexes. Advances in the field have now allowed for commercially available pooled sequences for greater reliability and efficiency, as described above, with less off-target effects. Their study used 50 nM staurosporine added to cells that had had proliferation halted with concanavalin A to induce apoptosis. We treated with 316 nM staurosporine without concanavalin A, which results in differentiation without obvious evidence of apoptosis (Frassetto et al., 2006
Gene silencing with siRNA is a methodology prone to artifact. While we cannot be sure that no off-target effects were seen, our use of 4 pooled siRNA duplex species is less likely to affect other genes, and we did not see knockdown of our actin control. Although we did not measure RNA levels, the near complete inhibition of immunodetectable SOD2 at the protein level by immunoblotting is evidence that substantial SOD2 knockdown was achieved. Other controls included pooled scrambled RNA that was used to account for the known toxic effects of siRNA, particularly at higher concentrations. These methods employed to achieve knockdown of the SOD2 gene have not been previously described in RGC-5 cells or RGCs, and our results demonstrate high transfection efficiency and reliable and consistent knockdown, with our small sample size yielding a relatively low standard error at 96 hours post-transfection. There was moderate loss of RGC-5 actin over time in both the SOD2 and scrambled knockdown. This is likely due to a combination of the effects of the transfection procedure, toxic effects of staurosporine, and the fact that neuronally differentiated RGC-5 cells were not supported with neurotrophic factors.
Brain-derived neurotrophic factor (BDNF) has been shown to increase survival of RGC-5 cells, and supplementation may result in increased viability (Harper et al., 2009
). Our finding of increased RGC-5 cell death when there was preferential knockdown of SOD2 beyond that of actin appears consistent with a mechanism of action for superoxide as a signal for neuronal cell death. Our previous studies ruled out neurotrophin deprivation as the cause of elevated superoxide after RGC axotomy because a combination of neurotrophins did not prevent increases in HEt-detectable superoxide (Lieven et al., 2006
). Therefore, it is unlikely that the introduction of neurotrophic factors would have affected superoxide production or SOD2 knockdown. When PEG-SOD was added to menadione treated cells, there was a small yet significant increase in cell viability. In theory this would have been expected to be even greater, but it is likely that PEG-SOD does not enter the mitochondrial matrix as effectively as other mitochondrial or cytoplasmic compartments.
In summary, we found that SOD2 knockdown in differentiated RGC-5 cells is associated with increased levels of superoxide and cell death. Reduced SOD2 expression creates higher concentrations of superoxide that are likely the cause of the observed decreased viability. When oxidative stress was induced with pharmacological treatments that increase superoxide levels, greater superoxide production and cell death were observed. These results provide a model for the study of cell-autonomous superoxide signaling in vitro
after optic retinal ganglion cell body or axonal injury, and also support prior studies showing that SOD2 is an endogenous scavenger for RGC survival in vivo (Qi et al., 2003
). Our data also establish the use of siRNA in staurosporine-differentiated RGC-5 cells as a method for studying neuron-specific mechanisms of cell death and survival.